Micellar ordered structure effects on high‐resolution CE‐SSCP using Pluronic triblock copolymer blends

Pluronic F108 block copolymers have shown a great promise to achieve the desirable high resolution in the conformation‐sensitive separation of ssDNA using CE‐SSCP. However, fundamental understanding of the structures and properties of Pluronic matrix affecting the resolution is still limited. Unlike conventional gel‐forming homopolymers, Pluronic F108 block copolymers are amphiphilic macromolecules consisting of poly(ethylene oxide)‐b‐poly(propylene oxide)‐b‐poly(ethylene oxide) triblock copolymers, which are capable of forming a highly ordered micellar structure in aqueous solution. In this study, we have performed a series of experiments by blending different types of Pluronic polymers to control the formation of micelles and to study the correlation between separation and rheological characteristics of Pluronic gels affecting the resolution of CE‐SSCP. Our experiments have been specifically designed to elucidate how the micellar structure affects the resolution of CE‐SSCP upon altering the size uniformity and constituent homogeneity of the micelles. Our results suggest that uniformly sized micelle packing is the primary structural feature of Pluronic gel matrix for the high‐resolution separation, while the size and constituent of the micelle themselves need to be considered as secondary factors.     

A novel pathogen detection system based on high‐resolution CE‐SSCP using a triblock copolymer matrix

Although CE‐SSCP analysis combined with 16S ribosomal RNA gene‐specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE‐SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE‐SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE‐SSCP analysis. A poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE‐SSCP analysis using the PEO‐PPO‐PEO triblock copolymer as the polymer matrix and four same‐sized DNA fragments of similar sequence content. Among 48 commercially available PEO‐PPO‐PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO₁₃₇PPO₄₃PEO₁₃₇ exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE‐SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO‐PPO‐PEO triblock copolymer may serve as an ideal matrix for high‐resolution CE‐SSCP analysis.     

A simple and precise diagnostic method for spinal muscular atrophy using a quantitative SNP analysis system

A simple and precise diagnostic method for spinal muscular atrophy (SMA) using high‐resolution CE‐based single‐strand conformation polymorphism (CE‐SSCP) was developed in this study. SMA is a common genetic disorder caused by an abnormality in the relative copy numbers of SMN1 and its centromeric copy SMN2, which differ only in two nucleotides, namely at exons 7 and 8. Therefore, the precise discrimination of the differences in sequence as well as their relative quantities is crucial for the diagnosis of SMA. Multiplex ligation‐dependent probe amplification and sequence‐sensitive DNA separation using hydroxyethyl cellulose and hydroxypropyl cellulose blended polymer matrix are currently the available methods used in the diagnosis of SMA. However, these methods are limited by their extended hybridization step and low resolution. In this study, the simultaneous discrimination of SMN exons 7 and 8 was successfully demonstrated using high‐resolution CE‐SSCP. Unlike the previously reported alternative method, single base differing amplicons were baseline‐separated because of its extraordinary resolution, thus providing accurate and precise quantification of each paralog.     

Triblock copolymer matrix‐based capillary electrophoretic microdevice for high‐resolution multiplex pathogen detection

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE‐SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer as a sieving matrix for CE‐SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO‐PPO‐PEO copolymers, 255‐bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO‐PPO‐PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan? gel. Due to enhanced dynamic coating and sieving ability, PEO‐PPO‐PEO copolymer displayed fourfold enhancement of resolving power in the CE‐SSCP to separate same‐sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high‐resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO‐PPO‐PEO triblock copolymer an excellent matrix in the CE‐SSCP analysis on the microdevice.     

High‐resolution pluronic‐filled microchip CE‐SSCP analysis system via channel width control

Although the resolution of CE‐SSCP has been significantly improved by using a poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO; Pluronic^®) triblock copolymer as a separation medium, CE‐SSCP on a microchip format is not widely applicable because their resolution is limited by short channel length. Therefore, a strategy to improve the resolution in channels of limited lengths is highly required for enabling microchip‐based CE‐SSCP. In this study, we developed a high‐resolution CE‐SSCP microchip system by controlling the width of the pluronic‐filled channel. We tested four different channel widths of 180, 240, 300, and 400 μm, and found that 300 μm showed the highest resolution in the separation of two pathogen specific markers. Potential applications of our method in various genetic analyses were also shown by using SNP markers for spinal muscular atrophy.     

Effect of temperature gradients on single-strand conformation polymorphism analysis in a capillary electrophoresis system using Pluronic polymer matrix

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35 °C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30 °C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix.     

A viscosity-tunable polymer for DNA separation by microchip electrophoresis

A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17–19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis. Figure Temperature dependence of the viscosity of 20% w/w Pluronic F127 solution in 1xTBE buffer. Dotted approximates resultant curve.     

Star-shaped polymers for DNA sequencing by capillary electrophoresis

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Cover Picture: Electrophoresis 21'2010

Issue no. 21 is a regular issue with "Emphasis on Nucleic Acids" and comprises 17 contributions distributed over 4 distinct parts. Part I is on nucleic acids and has 8 research articles on various aspects of nucleic acid research including an automated instrument for human STR identification, a high resolution chip‐CE assay for rapid detection of gene mutations and amplifications, a fluorescence‐based sequence‐specific primer PCR, pulsed field CE for RNA separation, methylated DNA immunoprecipitation sequencing, PCR‐SSCP, DNA methylation, and analysis of SNPs. Part II has 4 research articles dealing with studies on protein and proteomics analysis, e.g., microchip IEF, CE analysis of microheterogeneous glutelin subunits in rice, protein precipitation methods and prefractionation of plasma for proteomic analysis. Part III has 2 contributions that describe the separation of the enantiomers of DL‐isocitric acid by CE and the analysis of trace degradation products of S‐adenosylmethionine, which are decarboxylated diastereomers, by CE. The last 3 articles in this issue (Part IV) are on novel methodologies for facile interfacing of CE with MS, electrokinetic chromatography for the determination of contaminants and impurities of heparin samples, and particle electrophoresis. As such, issue no. 21 is packed with the latest developments and innovations in the field. Featured articles include: An automated instrument for Human STR identification: design, characterization, and experimental validation (doi: 10.1002/elps.201000305 )) Miniature flowing atmospheric‐pressure afterglow ion source for facile interfacing of capillary electrophoresis with mass spectrometry (doi: 10.1002/elps.201000350 )) Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.). (doi: 10.1002/elps.201000333 ))     

Single strand conformation polymorphism analysis of ras oncogene by capillary electrophoresis with laser-induced fluorescence detector

Single strand conformation polymorphism (SSCP) analysis of the N-ras oncogene was achieved by capillary electrophoresis with a laser-induced fluorescence detector (CE-LIF) using methylcellulose as a molecular sieving agent. The PCR-amplified N-ras oncogene, which is known to have a point mutation at codon 61 in the neuroblastoma, was investigated by CE-LIF combined with SSCP (SSCP-CE-LIF). A mixture of wild- and mutant-type single strand DNA fragments (103 bp) of the N-ras oncogene was separated by buffer solution containing 1.0% methylcellulose and 0.2 microM fluorescent dye (YO-PRO-1) at 25 degrees C. The SSCP-CE-LIF technique gave good resolution for wild- and mutant-type single strand DNA fragments with separation completed within 7 min. SSCP analysis using a CE system with a LIF detector was successfully applied to the detection of the one point mutation on the N-ras oncogene.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

Molecular recognition by chiral cationic micellar and micelle-like aggregates in electrokinetic capillary chromatography

We examined the enantiomer separation with micelles and a micelle-like polymer made with trimethylammonium-terminated surfactants all of whose hydrocarbon chains contain hydrogen bonding valinediamide moieties in electrokinetic chromatography (EKC). The surfactants used were 3-(N-dodecanoyl-L-valylamino)-propyltrimethylammonium bromide (surfactant 1) and 6-(N-nonanoyl-L-valylamino)hexyl-trimethylammonium bromide (surfactant 2); the micelle-like polymer was derived from 3-(N-10-undecenoyl-L-valyl)aminopropyltrimethylammonium bromide (surfactant 3). N-Acylamino acids and their isopropyl esters were separated with enantiomers with the same configuration as the chiral surfactant and which were retained to a greater extent than the counterparts in micelles. The micellar hydrophobic environment, in which amides function as hydrogen bonding sites with solutes, and ceased micellar kinetic association-dissociation with polymerization are discussed.     

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer-solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water-soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi-angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on-line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.     

Matrix conditioning for lengthened capillary DNA sequencing

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.     

Novel covalently coated diazoresin/polyvinyl alcohol capillary column for the analysis of proteins by capillary electrophoresis

A novel method for the preparation of covalently linked capillary coatings of PVA was demonstrated using photosensitive diazoresin (DR) as coupling agents. Layer‐by‐layer self‐assembly film of DR and PVA based on hydrogen bonding was first fabricated on the inner wall of capillary, then the hydrogen bonding was converted into covalent bonding after treatment with UV light through the unique photochemistry reaction of DR. The covalently bonded coatings suppressed basic protein adsorption on the inner surface of capillary, and thus a baseline separation of lysozyme, cytochrome c and BSA was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVA coatings, the covalently linked DR/PVA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of highly toxic and moisture‐sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make the covalently coated capillaries for CE.     

A molecularly imprinted polymer receptor for the enantiomeric recognition of amino acid hydantoins mimicking cooperative hydrogen bonds between nucleotide bases

Using acrylamide as hydrogen bonding functional monomer and (5R)-5-benzylhydantoin as template, a molecularly imprinted polymer was prepared in a polar solvent, which exhibited good enantiomeric recognition properties. The binding characteristics and selectivity of the polymer were evaluated by batch methods. Scatchard analysis showed that two classes of binding sites were produced in the polymer matrix and their dissociation constants were calculated to be 3.5 × 10-5mol/L and 4.3 ×10-4 mol/L, respectively, by utilizing the model of multiple independent classes of binding sites. These results were more reasonable than those obtained by Scatchard analysis , which was in agreement with the prediction of the binding characteristics of the polymer by exploring the effect of acrylamide on UV spectra of (5R)-5-benzylhydantoin. The substrate- and enantio-selectivity of the polymer was investigated. Finally, the study of effect of water on the chiral separation factor of the polymer further proved that the hy     

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low‐affinity DNA binders

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial‐filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low‐affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly‐dT) of different lengths (from 5 to 60‐mer) were investigated. It was shown that the size increase‐dependent random coil‐like conformation of Poly‐dT favorably acted on the apparent selectivity and resolution. The base‐unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single‐stranded to double‐stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single‐stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers.